Purpose:
Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in the elderly. The dry form is more common and accounts for about 85~90% of AMD patients in US, while Japanese AMD patients predominantly progress to wet-form or polypoidal choroidal vasculopathy (PCV). Recent studies have shown HTRA1, a serine protease gene, as major risk factor for wet form AMD. Furthermore, we reported that the Japanese typical wet form AMD patients showed significant association with ARMS2/HTRA1. The purpose of study is to elucidate the function of ARMS2-HTRA1 gene regulatory element in wet-form AMD patients.

Methods:
Human peripheral blood was obtained from patient with control and Wet-AMD. Genome DNA was extracted from peripheral blood samples and DNA sequenced using ABI 3130 Genetic analyzer. ARMS2/HTRA1 regulatory element activity was measured by luciferase assay. Double strand DNA probe was designed based on the wild-type and mutant sequence and Electrophoresis Mobility Shift Assay (EMSA) was performed. The same probe was used to isolate binding transcription factors and to determine the peptide sequence using liquid chromatography-mass spectrometry (LC-MS/MS). More, We also generated a transgenic (Tg) mouse, which overexpress mouse Htra1 and human ARMS2 in the entire body and observed the pathological change by fundus observation, fluorescein angiography, indocyanine green angiography and optical coherence tomography over 12 month after birth.

Results:
The regulatory element sequence experiment showed that a great number of AMD patients had specific indel mutation in 3.8 Kb upstream of HtrA1 gene. 2~3-fold increase of regulatory element activity was observed in indel HTRA1 regulatory element compared to control sequence. Furthermore, we detected indel specific binding factors using EMSA and LC-MS/MS. These results suggest that Htra1 gene expression is influenced by transcription factor specifically binding to this region. And more, using transgenic mice ubiquitously overexpressing mouse HTRA1 using the chicken actin promoter, continuous induction of HTRA1 in vivo was shown to lead to CNV, similar to wet AMD patients

Conclusions:
Human HTRA1 expression is enhanced by AMD specific indel mutation in the regulatory element region of HTRA1 gene. Specific transcription factor, which likely to be involved in this enhancement was isolated and peptide sequence determined.